Method for producing l-glutamic acid



United States Patent Office 3,511,752 Patented May 12, 1970 3,511,752 METHOD FOR PRODUCING L-GLUTAMIC ACID Katsunobu Tanaka and Kazuo Kimura, Machida-shi, Japan, assiguors to Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan, a corporation of Japan No Drawing. Continuation of application Ser. No. 656,011, July 26, 1967. This application July 17, 1968, Ser. No. 745,355

Int. Cl. C12d 13/06 US. Cl. 195-28 16 Claims ABSTRACT OF THE DISCLOSURE L-glutamic acid is produced by fermentation of a nutrient medium containing hydrocarbon as principal or sole assimilable carbon source together with a nitrogen source and inorganic salts, and is enhanced by the inclusion of acetic acid, or of an antibiotic, or of a surface active agent, or of a higher fatty acid, as an additional component, Microorganisms which have been found suitable for the fermentation are of the genera Coryne-bacterium, Arthrobacter, Brevibacterium and Micrococcus.

CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation application of copending application Ser. No. 656,011, filed on July 26, 1967, which is a continuation-in-part application of Ser. No. 433,478 filed on Feb. 17, 1965 both now abandoned.

BACKGROUND OF THE INVENTION It is well known to produce L-glutamic acid by fermentation, on an industrial scale, by culturing certain microorganisms on nutrient media containing carbohydrates as the carbon source. The carbohydrate substrates employed, however, present the disadvantage of being relatively expensive raw materials.

It is also known to produce L-gluta-mic acid by fermentation using microorganisms capable of converting a hydrocarbon, or a mixture of hydrocarbons, as a principal carbon source, when cultured on a nutrient medium containing such hydrocarbons. Although this has the advantage of employing cheaper raw materials, the quantity of L-glutamic acid produced is generally of the order of only several hundred gammas per ml. of the nutrient medium. In other words, the amount of amino acid produced is so minuscule, in reported cases of such prduction, that the amino acid thus produced has to be regarded as nothing more than a product of autolysis of the mieroorganism. In any event, such conversion with production of essentially no more than traces of amino acid can hardly be regarded as of significance in so far as the commercial production of glutamic acid is concerned.

ment of commercially feasible processes for the microbiological or fermentative production of L-glutamic acid from a readily accessible and very cheap source of carbon, namely hydrocarbon or a mixture of hydrocarbons.

The present invention is based onthe observation that microorganisms of the genera Corynebacterium, Arthrobacter, Brevibacteriu-m, and Micrococcus, are not only capable of fermentatively producing remarkable large amounts of L-glutamic acid in an aqueous nutrient medium containing hydrocarbon or hydrocarbon mixture as principal or sole assimilable carbon source and are therefore especially adapted for use in commercial scale operations, but that their hydrocarbon-conversion capacity is further considerably increased by the concomitant presence in the nutrient medium (fermentation liquor) of a conversion-enhancing additive, which may be acetic acid (as such or in the form of its alkali metal salt, e.g. sodium salt or potassium salt, or ammonium salt), or an antibiotic, or a surface-active agent, or a higher fatty acid. This enhancement of the conversion of hydrocarbon(s) into L-glutamic acid correspondingly increases the significance of the process as commercially feasible and industrially practicable. A further characteristic advantage of the invention is that 'L-glutamic acid is essentially the sole conversion product in the liquid culturing medium.

The conversionenhancing agent may itself function as assimilable carbon source for the conversion under consideration. However, the conversion realized by the concomitant employment of hydrocarbon(s) and, for example, acetic acid or salt thereof, is considerably in excess of the sum of the conversions into L-glutamic acid realizable from the said two carbon source individually.

The conversion-enhancing additive, which is preferably used in small amount in comparison to the hydrocarbon(s) constituting the principal and low-cost carbon source, not only increases the efliciency of the hydrocarbon(s) employed, but results in suppression of formation, as by-products, of undesired amino acidsi.e. amino acids other than L-glutamic acid-such by-product formation of by-product amino acids being an undesirable incident when saccharidic materials are employed as assimilable carbon source.

As for the hydrocarbons used as assimilable carbon source in the process of the present invention, aliphatic hydrocarbons with 10 to 20 carbon atoms in the molecule can be used. However, best yields of L-glutamic acid are obtained with normal paraffinswith 10 to 18 carbon atoms. Thus, useful hydrocarbons for the purposes of the present invention comprise decane, undecane, dodeeane, tridecane,.tetradecane, pentadecane, hexadecaue, heptadecane, octadecane, nonadecane and eicosane, as the normal compounds or in the iso.forms, individually or in admixtures sueh as are frequently obtained in practice. However, the normal paraflins: n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane,- n-hexadecane, n-heptadeeane and n-octadecane, severally or in ad mixture, e.g. in the form of commercial mixtures, give superior results.

DESCRIPTION OF SPECIFIC ILLUSTRATIVE FEATURES AND PROCEDURES The following table exemplifies the production of L- glutamic acid according to this invention and illustrates in particular the synergistic elfect of the presence of acetic acid or salt thereof in enhancing the conversion of the hydrocarbon used as principal carbon source:

TABLE 1* Oulturing Gulturing culturing medium A; medium B; medium production production production amount of amount of amount of L-glutumlc L-glutnuiic L-glutunuc acid (yield) acid (yield) acid (yield) Name of strain mgJml. mgJml. mgJml.

Arthroizzcteratmplemglo.3gb (AtTCCbIIQo. 1 5 4.6 (9.2%) 3.8 (3.8%) 18.8 (31%) 00 na acterium h room 0 as us 0.

(Circe No.1559 4.2 (8.4%) 4.5 4. 5%) 17.9 (30%) All percentages are by weight. Norm: Carbon source of culturing medium A 5% of undecu ie. Carbon source of culturing medium B: 1% of sodium acetate (as acetic acid Ciirbon source of culturing medium C: 5% of un'decanc and 1% of sodium acetate (as acetic acid). Further constitution Example 1 of nutrient medium: Percent by wt.

Remainder water; v pH 7.0.

Culturing conditions.-To three separate nutrient or fermentation mediums constituted as indicated, one of the carbon sources A, B and C was added, the medium then inoculated with Arthrobacter simplex No. 3151 and subjected to shaking culture at 30 C. for 72 hours with 120 reciprocations per minute.

pH of the medium was controlled by the addition of (NH CO and the value of 6.0 to 8.0 was maintained. The procedure was thereafter repeated with Corynebacterium hydrocarboclastus No. 2438.

It is clear from Table 1 that the production yield of L-glutarnic acid from hydrocarbon is exceedingly increased in every case by the addition of, ,efiectively, acetic acid.

In practicing the process of this invention, the fermentation medium may contain, in addition to the hydrocarbon and conversion-enhancing additive as carbon sources, con ventional nitrogen sources e.g. ammonium sa.t such as ammonium sulfate, ammonium chloride or the like or urea, also conventionally-employed inorganic salts and 1 some usual organic-nutrient. As organic nutrient, peptone, corn steep liquor, meat extract, yeast extract, hydrolysis product of soy bean meal or the like is useful. The addi- Q tion of meat extract is particularly effective in the production of L-g1utamic acid. Instead of these organic nutrients, it is possible to produce L-glutamic acid with F high production yield by the addition of various vitamins,

amino acids, organic nucleic acid base or the like. During the course of fermentation, aerobic culturing condition is required and it is important to maintain pH at 5.0 to 9.0, particularly at 6.0 to 8.0, by the addition of e.g. ammonium salt, ammonia or urea during the culturing period.

In order that those skilled in the art may more fully i understand the nature of the invention and the method of carrying it out, the following ilustrative examples of l presently preferred embodiments are given. In these examples, the relationship between parts by weight and parts by volume is the same as that between grams and milliliters.

Two hundred parts by volume of a fermentation medium containing Percent by wt.

Remainder water; pH 7.0.

are introduced into a receptacle having a capacity of 2000 parts by volume. In this medium, the strain of hydrocarbon-converting microorganism, Arthrobacter simplex No. 3151 (ATCC No. 15799) is inoculated and subjected to shaking cu'ture, under aerobic conditions at 30 C. for 72 hours at 220 reciprocations per minute. The accumulation of L-glutamic acid reaches 20.0 milligrams per milliliter.

During the cultivation, the pH is controlled and maintained at 6.0 to 8.0 by the addition of (NH CO when necessary.

Upon completion of the cultivation, the L-glutamic acid is removed from the fermentation broth by passing the latter through cation exchange resin, in per se known mariner, whereupon the L-glutamic acid is adsorbed. The L-glutamic acid is then eluted with di ute aqueous ammonia, the obtained solution concentrated and then cooled. Crystals of L-glutamic acid separate, are isolated and recrystallized. The recovery or harvesting of the L-glutamic acid from the fermentation liquor is per se no part of the present invention, and may be carried out in any manner known in prior fermentative processes 'for the production of L-glutamic acid. (Any other known procedure for recovering L-glutamic acid from a fermentation medium in which it was produced, can also be employed.)

About 3.2 parts of recrystallized L-glutamic acid are obtained.

Example 2 Ten thousand parts by volume of culture medium of the following composition were prepared:

Percent by wt. KH P0 0.1 Na HPO 12H O 0.1 MgSO -7H 0 0.1 FeSO -7H 0 0.02 M11804 Meat extract 0.1 (NH S0 1.0 Ammonium acetate (as acetic acid) 1.0 Phenol red 0.001 Remainder water; pH 7.0.

and sterilized by heat in a fermenter of 20,000 parts by volume capacity.

Corynebacterium hydrocarboclastus No. 2438 (ATCC No. 15592) is precultured by aerobic shaking culture in a liquid bouillon medium containing 1% by weight of kerosene (petroleum distillation fraction consisting essentially of a mixture of n-C to n-C parafiin hydrocarbons; B.P. about 200300 C.) for 24 hours.

The thus-obtained preculture is introduced into the Corynebacteriumhydrocarboclastus No. 2438 (ATCC No. 15592) is precultured by aerobic shaking culture in a liquid bouillon medium containing 1% by weight of kerosene, for 24 hours.

The thus-obtained preculture is introduced into the sterilized culture medium in the aforesaid fermenter, in a proportion of about 5% by weight, and cultivation is carried out for 96 hours with stirring at about 30 C. at about 300 revolutions per minute while passing into the sterilized culture medium in the aforesaid fermenter, in fermentation liquor one liter of sterilized air per liter of a proportion of about 5% by volume, and culturing is medium per minute. carried out at 30 C. for 72 hours with stirring at about At the start of the cultivation period, 500 parts by 300 revolutions per minute while passing in one liter of volume of a mixture consisting of C11, C C13, C C bacteria-free air per liter of medium per minute. C C and C n-paraffins is added, and the pH is main- Starting at the twelfth hour of such culturing in the 15 tained at 6.8 to 8.0 by introducing ammonia gas during said fermenter, kerosene is added to the medium at the the said period, and Penicillin G was added 12 hours after rate of about 1.2 percent by weight in the course of every the beginning of fermentation to maintain 50 units/ ml. 12 hours in such manner that a total of 5% by weight concentration thereof in the medium. will have been added by the end of the culture period. The production of L-glutamic acid at the end of the During the culturing period (NH CO is added, simul- 20 fermentation amounted to 10.5 milligrams per milliliter. taneously with the addition of the kerosene, to assure maintenance of the pH of the fermentation liquor at 6.8. Example 5 The production of L-glutamic acid at the end of the 0 fermentation amounts to 15.1 milligrams per milliliter. Q Pgiutamlc obtalfled y addlt'lon of 1 3 various antibiotics m fermentation using microorganisms Exam? 6 capable of fermentatively converting hydrocarbons to L- The procedure according to Example 1 was repeated, glutamic acid are Shown in T b e except that the 0.1% of meat extract was replaced by The fermentation medium utilized was as follows: 0.3% of corn steep liquor, the 1% of sodium acetate (as acetic acid) was replaced by 1% of arn- Percent y monium acetate (as acetic acid), and Corynebacterium 2 4 hydrocarboclastus No. 24322 (ATCC No. 15592 was z 4- 2 used as the converting microorganism in lieu of Arthroe' z bacter simplex No. 3151; in addition 50 units of Penicillin 2 G per milliliter were added at the 6th hour of culture. In 4' 2Q 0-002 other respects, the same culturing procedure was followed Steep hquor as in Example 1. From 200 parts by volume of culturing NH4N08 liquor, about 2.2 parts by weight of L-glutamic acid were mixture (Equlvalent "fixture of obtained. CTCM) 40 Phenol red 0.001 Example 4 Remainder water; pH 7 .0.

Ten thousand parts by volume of culture medium of Twenty (20) ml. of the above-specified fermentation the following composmon were prepared medium were placed in 250 ml. conical flasks.

Percent by wt. 5 The various strains of hydrocarbon-converting micro- KH PO 0.1 organisms are pre-cultured in the medium containing KHPO 0.1 0.25% yeast extract, 0.5% bouillon, 0.5% peptone, 0.25 MgSO -7H O 0.05 NaCl, for 24 hours. 4' 2 The above precultured strains were inoculated into 5 4 2 0-002 the aforesaid fermentation medium and subjected to (gfiii g shaking-culture at 30 C. for 12 hours. During the culti- 'g;"gags- 7 vation, the pH is controlled and maintained at 6.5 to p u 7.0 by the addition of (NH CO and urea when necesand stenhzed by heat in a fermenter of 20,000 parts by sary. Dosage and time of addition of the antibiotics are volume capacity. 0 shown in Table 2.

TABLE 2 Time of addition of antibiotics after beginning Amounts of of L- lutnmic fermentation aci formed Name of microorganisms used Antibiotics added Dosage rs.) (mgJmL) Arthrobacter parafii'neus:

AICC Number 15591.--. Penicillin G so /m1. 24 12, 8 Do Kephrine 'y/ml. 24 15.2 Do... Baeitracm 100 'y/ml. 24 10. 2 Do- 100 'y/ml. 24 9.6 30. 50 -y/ml. 24 11.0 Do- 50 'y/ml. 30 10.1 o. 100 'ylml. 24 8. 2 Do- Stre tornycin eomplex..;- 50 -y/ml. 24 9.5 Do-.. Cyc oserine 100 'y/m 24 10.2 3 (C S a 100 'y/ml. 24 13.0 Brevgig cercidfiiceigiiiiltgz ggch vni um er P Hill G 1. 5:"-

it n ii iii D03: (Control) 3.1: iii

TABLE Zr-Continued Time of addition of antibiotics in ginii l A ts f a rig menu 0t L-glutarnie fermentation aeld formed N amo of mlcro-organlsms used Antibiotics added Dosage (hrs) (mg/ml.) Arthrobacter hudrocarboglutamicus:

ATCC Number 15583 Penicillin G 50 u/ml. 24 12.4 -D o- Kophrlno 100 /1111. 24 11.5 Do... Spiramycin 50 'ylml. 24 10.8 Do- (Control) i 1.6 Arthrubacter roseoparaflineus: 1 ATS!) Number 15584 Penicillin G 50 Iml. 24 8.9 I o (Control) 2.3 Brevibacterium keto-glutamlcum:

ATCC Number 15587 Penicillin G 50 1/1111. 24 12.7 Do (Control) 2. 5 Arthrobacter paraflineua ATCG Number 15590 Penloillm G 1/1111. 24 14.5 Do (Control) 21 1.7 Corumbacterium hydrocar cl us TCO Number 15592- Penicillin G. 50 /ml. 24 11.2 v o (Control). 7 2. 1 Mfcrococcus araflmolyticus:

umber 15581.--. Penicillin G- 4/1111. 24 8. 0 o (Gontrol).. 0. 5 Mz'crococcua parafinolytzcua:

' ATCC Number 15589 Penielllm G- 5014/1111. 24 5.9 Do (Control) 0.2

Example 6 30 Example 7 1 carbon, are shown in Table 3. The same culturing pro- By the addition of various surface active agents or higher fatty acids, at the beginning of cultivation, amounts of glutamic acid formed in the culture medium by microorganisms capable of fermentatively converting hydro- 35 cedure was followed as in Example 5, with the exception that the content of corn steep liquor in the medium was 0.05%, and no antibiotic was added.

By the addition of various surface active agents or higher fatty acids 24 hours after the beginning of fermentation, amounts of glutamic acid formed in the culture medium by microorganisms capable of fermentatively converting hydrocarbon are shown in Table 4. The same culturing procedure was followed as in Example 5, with the exception that the content of corn steep liquor in the medium was 0.1%, and no antibiotic was added.

TABLE 3 Amounts of a r am orme Microorganisms used Additional components added Dosage Time of addition (mg/ml.) Arthrobacter paraffineus: I ATOC Number 15591 Nonion 0T-221 (also known as lween 80, /1111. At the beginning of cultivatlon 1s. 2

vlvlhigh is polyoxyethylene-sorbitan monoee Do Nonion LP-20R (also known as Span 20 mow/m1. -.-.do 17.5

which is sorbitan mono-laureate) Do. Nonion ST-221 (also known as Tween 60, 100 /1111. -....do 19.6

ughich is polyoxyethylene sorbitan monos eara Do 112101836 (alkyl betalne-ampholytlc surlog /m1. ...-.do 18, 0

ac n Do 1 7/111!- -...-do 18.2 Do (Control). 09 15. Brevibacterium ketoglutamicum:

ATCC Number 15588 Nonlon 0T-221. 17. Do (00:11:01)"... 14. Arthrobucter porafiineusz .A'ICC Number 15590. Nonlon ST-221 19, Do- Gaprylic acid.- 17. Do- Laurie acid 18. Do. Myristic acld. 19 Do (Control) 13. Brevibacterium keto-glutamicum:

ATCC Number 15587 Stearic acid Do Oleic acid-..

(Control) Do Arthrobacter hydrocarbo-glutamicus:

AT 0 Number 15583 Nonion LP-20R. lolly/ml Do (Control) Arthrobacter roseo arafimcus:

ATCC Num or 15584 Nonlon LIP-20R 100-ylml.

Do (Control) Carynebucterium hudrocarbo-clastus:

ATCC Number 15692.. Nonion OT-221 100 /1111.

Do (Control)- Micrococcus paraffmolyticu ATCG Number 15582 Nonlon OT-221 IOO-yIml.

Do (Control) Do- 100'yIml.

TABLE 4 Time of addition after Yield of beginning L-glutumic fermentation ac Microorganisms used Additional components added Dosage (hrs) (mg/ml.)

Arthrobacter purajfinzus:

A'IOO Number 15591 Nymeen S215 (polyoxyethylene alkylamine, a cationic sur- 0.5 mg./ml. 24 18. 2 Do Softer #601 (alkyl betaine, an anionic sufactant) 0.5 mgJml. 24 18. 0 Do T acid 0.5 mgJml. 24 18. 9 D Abietic i 0.5 mg./ml. 24 19. 0 B b Donnie? (Control) 2. 0

ram acterium to utamicum:

ATTC Numb r 15587 Catiogen H... 0 5 mg./ml 24 17. 5 D0 Linoleic acid.-. 0 5 mg./m1 24 16. 8 Abietic aci 0 6 mg./ml 24 17. 7 (Contro1) 1.8

Catio en H 0.5 mgJml. 24 18. 9 Linol ic aci 0.5 mg./ 24 18.6 Abietic acid 0.5 mg./ml. 24 17. 1 (Control)- 1. 6

What is claimed is:

1. In amethod for producing L-glutamic acid from hydrocarbon by fermentation of an aqueous nutrient medium containing a carbon source and a micro-organism capable of converting paraflinic hydrocarbon into L-glutamic acid, the improvement according to which the carbon source is constituted by a major proportion of parafiinic hydrocarbon, and a minor proportion of a conversion-enhancing agent whereby the L-glutamic acid is enhanced, said paraflinic hydrocarbon containing from ten to twenty carbon atoms, said conversion-enhancing agent selected from the group consisting of acetic acid, and an acetate.

2. A method according to claim 1, wherein the conversion-enhancing agent includes an antibiotic.

3. A method according to claim 2, wherein the antibiotic comprises penicillin.

4. A method according to claim 1, wherein the conversion-enhancing agent includes a surface-active agent.

5. A method according to claim 1, wherein the conversion-enhancing agent includes a higher fatty acid.

6. In a method for producing L-glutamic acid from hydrocarbon by fermentation of an aqueous nutrient medium containing a carbon source and a microorganism capable of converting paratfinic hydrocarbon .into L-glutamic acid, the improvement according to which the carbon source is constituted by a major proportion of a parafiinic hydrocarbon, and a minor proportion of a conversion enhancing agent whereby the L-glutamic acid production is enhanced, said paraflinic hydrocarbon containing from ten to twenty carbon atoms, said conversionenhancing agent selected from the group consisting of acetic acid and an acetate, and said microorganism is selected from a group of genera consisting of Corynebacterium, Arthrobacter, Brevibacterium, and Micrococcus.

7. A method according to claim 6, wherein the microorganism is selected from the group consisting of Arthrobacter parafiineus, Arthrobacter hydrocarboglutamicus, Arthrobacte'r roseoparafiinus, Arthrobacter simplex, Brevibacterium ketoglutamicum, Corynebacterium hydrocarboclastus, and Micracaccus parafiinolyticus.

8. A method according to claim 6, wherein the microorganism is selected from the group consisting of Arthrobacter simplex (ATCC No. 15799), Corynebacterium hydrocarboclastus (ATCC No. 15592), and Arthrobacter parafiineus (ATCC No. 15591).

9. A method according to claim 1, wherein the conversion-enhancing agent is in the fermentation medium at the beginning of the fermentation.

10. A method according to claim 1, wherein the conversion-enhancing agent is added to the fermentation medium while the fermentation is continuing.

11. A method according to claim 6 wherein the conversion-enhancing agent includes an antibiotic.

12. A method according to claim 6 wherein the conversion-enhancing agent includes a surface-active agent.

13. A method according to claim 6 wherein the conversion-enhancing agent includes a fatty acid.

14. A method according to claim 7 wherein the conversion-enhancing agent includes an antibiotic.

15. A method according to claim 7 wherein the conversion-enhancing agent includes a surface-active agent.

16. A method according to claim 7 wherein the conversion-enhancing agent includes a fatty acid.

References Cited UNITED STATES PATENTS 3,080,297 3/1963 Phillips et a1. 47 3,222,258 12/1965 Iizuka et al 195-29 3,227,625 1/ 1966 Phillips 195-30 3,326,775 6/ 1967 Miescher 19547 3,313,709 4/1967 Phillips 195-28 OTHER REFERENCES Iguchi et al.: Agr. Biol. Chem. (Japan), vol. 29, No. 6, pp. 589-90, 1965.

Chemical Abstracts 64, 11S89f (1966). Chemical Abstracts 66, 54265W (1967). Chemical Abstracts 66, 64388c (1967).

LIONEL M. SHAPIRO, Primary Examiner U.S. Cl. X.R. 195-114 

